基质辅助激光解析电离飞行时间质谱分析细菌方法的规范化研究
摘要
目的：
基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionization time of flight mass spectrometry，MALDI TOF MS)是最近发展起来的一种分析化学技术，能提供各具特征的细菌质量指纹图谱，实现检测和鉴定的目的。本研究通过对MALDI TOF MS分析细菌的方法进行系统的研究，阐明影响MALDI分析的重要实验因素的作用，并建立MALDI TOF MS分析细菌的标准实验方法，从而实现不同菌种和同一种内不同菌株的鉴定，为建立炭疽芽孢杆菌和鼠疫耶尔森氏菌的质量图谱数据库奠定基础，从而能够快速准确地检测和鉴定我国不同来源的炭疽芽孢杆菌和鼠疫耶尔森氏菌，在生物恐怖或传染性疾病爆发时，作为微生物法医学的一种重要技术，快速、准确地鉴定细菌，并追踪其来源。
方法：
根据研究目的，选择以下菌株进行试验：革兰氏阳性菌包括含芽孢的炭疽芽胞杆菌和不含芽孢的金黄色葡萄球菌；革兰氏阴性菌包括鼠疫耶尔森氏菌，大肠杆菌和胞外多糖含量高的洋葱伯克霍尔德氏菌；同一属内不同种的细菌(伯克霍尔德氏菌属的7个种)；同一种内的不同菌株(11株大肠杆菌和20株金黄色葡萄球菌)。在试验中，以炭疽芽孢杆菌和鼠疫耶尔森氏菌为目标菌，试验了不同的样品处理方法包括直接分析，溶剂处理(单一溶剂处理和组合溶剂处理)和酶处理(溶菌酶和胰蛋白酶)，及不同的实验因素包括基质、基质溶剂、处理用溶剂、菌体的量、点样方法，通过MALDI TOF MS分析提供的细菌各种成分的质量信息，应用Microbelynx软件进行比较分析，确定标准的实验方法和影响分析结果的重要实验因素。
在标准实验方法建立后，对方法的重复性包括同一样品的重复性和批间样品的重复性，及方法的可行性包括安全性和稳定性进行了系统的试验，从而能够应用于分析我国不同来源的炭疽芽胞杆菌和鼠疫耶尔森氏菌。
结果：
本研究通过MALDI TOF MS分析细菌方法的研究，确定了影响分析结果的重要实验因素，建立了一种用于MALDI TOF MS的标准实验方法，具体的实验
一一一一一一遭燮鲤鲤巡壑通竺型暨
方案为4 mg的细菌依次采用。，1%TFA和氯仿:甲醇(l:l)处理后，沉淀的菌体
重悬于0.1%TFA中，改进的干滴法点样。基质溶液为CHCA溶于乙睛:甲醇:水
(l:l:l)，含有0.1%甲酸和0.01 M 18一冠一6。
应用此方法，在m/z 2 000一10 000范围内根据各自的特征图谱可区分不同特性
的细菌(不同属内的细菌)包括炭疽芽抱杆菌、金黄色葡萄球菌、鼠疫耶尔森氏
菌、大肠杆菌和洋葱伯克霍尔德氏菌;伯克霍尔德氏菌属的不同菌种;大肠杆菌
和金黄色葡萄球菌的不同菌株。
同一样品和批间样品的重复性较好;安全性试验中，鼠疫耶尔森氏菌采用建
立的标准实验方法处理后即能完全灭活，但未能实现炭疽芽抱杆菌的灭活目的;
稳定性实验中，样品处理后放置7天，能获得与样品处理后立即分析较为一致的
结果。
结论:
本研究完成了MALDI TOF MS分析细菌方法的标准化，阐明了影响MALDI
分析细菌的重要实验因素，建立了一种 MALDI分析细菌的标准实验方法，分析
过程简单、快速、重复性较好。该方法能提供较多的质量信息区分不同特性的细
菌(不同属内的细菌)包括含芽抱或不含芽抱的革兰氏阳性菌，及革兰氏阴性菌
包括胞外多糖含量高和低的细菌，同一属内的不同菌种和同一种内的不同菌株。
安全性试验和稳定性试验显示，采用标准方法处理细菌后，鼠疫耶尔森氏菌即能
完全灭活，在7天内分析能得到稳定的实验结果;炭疽芽抱杆菌的灭活还需进一
步试验，在7天内分析也能得到稳定的实验结果。基于建立的标准实验方法能够
建立强大的质谱数据库系统，通过快速搜索实现细菌的鉴定，在微生物法医学的
追踪鉴定中发挥重要作用。
Aim:
MALDI TOF MS is a recently developed analytical chemistry technology. For bacterial detecting and identifying, MALDI TOF MS could provide mass spectra containing different biomarkers specific to various bacteria. The aim of this research is to find out the experimental parameters that affecting the results when analyzing bacteria by MALDI TOF MS, and to set up a general sample preparation protocol for establishing mass database of Bacillus anthracis and Yersinia pestis. It will play an important role in rapid identification of bacteria, and tracing its origin in the scenario of bioterrorism and outbreak of infectious disease for microbial forensic analysis. Methods:
As for the universalness of sample preparation protocol for bacterial analysis by MALDI TOF MS, bacteria being investigated included: gram-positive bacteria including B. anthracis (spore-producing) and Staphylococcus aureus (non-spore-producing ); gram-negative bacteria such as Y. pestis, Escherichia coli and Burkholderia cepacia with high extracellular polysaccharides; different species of the same genus ( 7 species of Burkholderia ); different strains of the same species ( 20 strains of S. aureus and 11 strains of E. coli). The present study mainly employs three different sample preparation protocols: bacteria directly analyzing, treating bacteria by solvent (single treatment and combined treatment) and enzyme (lysozyme and trypsin). At the same time, different experimental factors such as matrix, solvent of matrix, solvent system used, bacterial quantities, sample application method, were investigated. The role of different factors in sample preparation was illuminated. By using the above protocols, spect
ra acquired by MALDI TOF MS with mass information will be selected for evaluating the ability to differentiate bacteria of different origins.
The reproducibility of the established protocol was evaluated by repeating analysis of same sample and different batches of culture. In addition, safety and
stability of the protocol were tested in order to analyze different virulent strains of B.
anthracis and Y. pestis deposited in other institutes outside Beijing.
Results:
In the present research, bacterial analysis was studied systematically through testing different sample preparation protocols and experimental factors by MALDI TOF MS. A universal sample preparation protocol for MALDI TOF MS was established. The protocol is listed as follows. Bacterial cells (4 mg) were washed by 0.1% TFA and chloroform: methanol (1:1) in turn and the pellet was applied to the plate using improved dried droplet method. Matrix CHCA was prepared in ACN: methanol: water (1:1:1) with 0.1 % formic acid and 0.01 M 18-crown-6.
This protocol could be used to differentiate B. anthracis, S. aureus, E. coli, Y. pestis and B. cepacia; different species of Burkholderia and different strains of E. coli and 5. aureus according to the typical peak profiles in mass range from 2 000 to 10 000 Da, respectively.
The reproducibility was proved perfect using this protocol. Y. pestis was inactivated entirely using established protocol, while B. anthracis was not killed even using additional methods. The results acquired from samples of B. anthracis and Y. pestis, which placed for 7 d after treatment using the established method, is well consistent with results obtained immediately after treatment. Conclusions:
Protocols of bacterial analysis by MALDI TOF MS were described systematically. A novel sample preparation protocol was developed for rapid and accurate analysis of bacteria by MALDI TOF MS. This protocol was simple, rapid and easy to perform with excellent reproducibility. It can product enough peaks to identify both gram-positive bacteria, including spore-producing bacteria (B. anthracis) and non-spore-producing bacteria (S. aureus), and gram-negative bacteria such as Y. pestis, E. coli and B. cepacia with high content of extracellular polysaccharides. This protocol was also applied to analyze different species of the same genus and different strains of the same species. Th
基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption ionization time of flight mass spectrometry，MALDI TOF MS)是最近发展起来的一种分析化学技术，能提供各具特征的细菌质量指纹图谱，实现检测和鉴定的目的。本研究通过对MALDI TOF MS分析细菌的方法进行系统的研究，阐明影响MALDI分析的重要实验因素的作用，并建立MALDI TOF MS分析细菌的标准实验方法，从而实现不同菌种和同一种内不同菌株的鉴定，为建立炭疽芽孢杆菌和鼠疫耶尔森氏菌的质量图谱数据库奠定基础，从而能够快速准确地检测和鉴定我国不同来源的炭疽芽孢杆菌和鼠疫耶尔森氏菌，在生物恐怖或传染性疾病爆发时，作为微生物法医学的一种重要技术，快速、准确地鉴定细菌，并追踪其来源。
方法：
根据研究目的，选择以下菌株进行试验：革兰氏阳性菌包括含芽孢的炭疽芽胞杆菌和不含芽孢的金黄色葡萄球菌；革兰氏阴性菌包括鼠疫耶尔森氏菌，大肠杆菌和胞外多糖含量高的洋葱伯克霍尔德氏菌；同一属内不同种的细菌(伯克霍尔德氏菌属的7个种)；同一种内的不同菌株(11株大肠杆菌和20株金黄色葡萄球菌)。在试验中，以炭疽芽孢杆菌和鼠疫耶尔森氏菌为目标菌，试验了不同的样品处理方法包括直接分析，溶剂处理(单一溶剂处理和组合溶剂处理)和酶处理(溶菌酶和胰蛋白酶)，及不同的实验因素包括基质、基质溶剂、处理用溶剂、菌体的量、点样方法，通过MALDI TOF MS分析提供的细菌各种成分的质量信息，应用Microbelynx软件进行比较分析，确定标准的实验方法和影响分析结果的重要实验因素。
在标准实验方法建立后，对方法的重复性包括同一样品的重复性和批间样品的重复性，及方法的可行性包括安全性和稳定性进行了系统的试验，从而能够应用于分析我国不同来源的炭疽芽胞杆菌和鼠疫耶尔森氏菌。
结果：
本研究通过MALDI TOF MS分析细菌方法的研究，确定了影响分析结果的重要实验因素，建立了一种用于MALDI TOF MS的标准实验方法，具体的实验
一一一一一一遭燮鲤鲤巡壑通竺型暨
方案为4 mg的细菌依次采用。，1%TFA和氯仿:甲醇(l:l)处理后，沉淀的菌体
重悬于0.1%TFA中，改进的干滴法点样。基质溶液为CHCA溶于乙睛:甲醇:水
(l:l:l)，含有0.1%甲酸和0.01 M 18一冠一6。
应用此方法，在m/z 2 000一10 000范围内根据各自的特征图谱可区分不同特性
的细菌(不同属内的细菌)包括炭疽芽抱杆菌、金黄色葡萄球菌、鼠疫耶尔森氏
菌、大肠杆菌和洋葱伯克霍尔德氏菌;伯克霍尔德氏菌属的不同菌种;大肠杆菌
和金黄色葡萄球菌的不同菌株。
同一样品和批间样品的重复性较好;安全性试验中，鼠疫耶尔森氏菌采用建
立的标准实验方法处理后即能完全灭活，但未能实现炭疽芽抱杆菌的灭活目的;
稳定性实验中，样品处理后放置7天，能获得与样品处理后立即分析较为一致的
结果。
结论:
本研究完成了MALDI TOF MS分析细菌方法的标准化，阐明了影响MALDI
分析细菌的重要实验因素，建立了一种 MALDI分析细菌的标准实验方法，分析
过程简单、快速、重复性较好。该方法能提供较多的质量信息区分不同特性的细
菌(不同属内的细菌)包括含芽抱或不含芽抱的革兰氏阳性菌，及革兰氏阴性菌
包括胞外多糖含量高和低的细菌，同一属内的不同菌种和同一种内的不同菌株。
安全性试验和稳定性试验显示，采用标准方法处理细菌后，鼠疫耶尔森氏菌即能
完全灭活，在7天内分析能得到稳定的实验结果;炭疽芽抱杆菌的灭活还需进一
步试验，在7天内分析也能得到稳定的实验结果。基于建立的标准实验方法能够
建立强大的质谱数据库系统，通过快速搜索实现细菌的鉴定，在微生物法医学的
追踪鉴定中发挥重要作用。
Aim:
MALDI TOF MS is a recently developed analytical chemistry technology. For bacterial detecting and identifying, MALDI TOF MS could provide mass spectra containing different biomarkers specific to various bacteria. The aim of this research is to find out the experimental parameters that affecting the results when analyzing bacteria by MALDI TOF MS, and to set up a general sample preparation protocol for establishing mass database of Bacillus anthracis and Yersinia pestis. It will play an important role in rapid identification of bacteria, and tracing its origin in the scenario of bioterrorism and outbreak of infectious disease for microbial forensic analysis. Methods:
As for the universalness of sample preparation protocol for bacterial analysis by MALDI TOF MS, bacteria being investigated included: gram-positive bacteria including B. anthracis (spore-producing) and Staphylococcus aureus (non-spore-producing ); gram-negative bacteria such as Y. pestis, Escherichia coli and Burkholderia cepacia with high extracellular polysaccharides; different species of the same genus ( 7 species of Burkholderia ); different strains of the same species ( 20 strains of S. aureus and 11 strains of E. coli). The present study mainly employs three different sample preparation protocols: bacteria directly analyzing, treating bacteria by solvent (single treatment and combined treatment) and enzyme (lysozyme and trypsin). At the same time, different experimental factors such as matrix, solvent of matrix, solvent system used, bacterial quantities, sample application method, were investigated. The role of different factors in sample preparation was illuminated. By using the above protocols, spect
ra acquired by MALDI TOF MS with mass information will be selected for evaluating the ability to differentiate bacteria of different origins.
The reproducibility of the established protocol was evaluated by repeating analysis of same sample and different batches of culture. In addition, safety and
stability of the protocol were tested in order to analyze different virulent strains of B.
anthracis and Y. pestis deposited in other institutes outside Beijing.
Results:
In the present research, bacterial analysis was studied systematically through testing different sample preparation protocols and experimental factors by MALDI TOF MS. A universal sample preparation protocol for MALDI TOF MS was established. The protocol is listed as follows. Bacterial cells (4 mg) were washed by 0.1% TFA and chloroform: methanol (1:1) in turn and the pellet was applied to the plate using improved dried droplet method. Matrix CHCA was prepared in ACN: methanol: water (1:1:1) with 0.1 % formic acid and 0.01 M 18-crown-6.
This protocol could be used to differentiate B. anthracis, S. aureus, E. coli, Y. pestis and B. cepacia; different species of Burkholderia and different strains of E. coli and 5. aureus according to the typical peak profiles in mass range from 2 000 to 10 000 Da, respectively.
The reproducibility was proved perfect using this protocol. Y. pestis was inactivated entirely using established protocol, while B. anthracis was not killed even using additional methods. The results acquired from samples of B. anthracis and Y. pestis, which placed for 7 d after treatment using the established method, is well consistent with results obtained immediately after treatment. Conclusions:
Protocols of bacterial analysis by MALDI TOF MS were described systematically. A novel sample preparation protocol was developed for rapid and accurate analysis of bacteria by MALDI TOF MS. This protocol was simple, rapid and easy to perform with excellent reproducibility. It can product enough peaks to identify both gram-positive bacteria, including spore-producing bacteria (B. anthracis) and non-spore-producing bacteria (S. aureus), and gram-negative bacteria such as Y. pestis, E. coli and B. cepacia with high content of extracellular polysaccharides. This protocol was also applied to analyze different species of the same genus and different strains of the same species. Th
引文
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